Toll-like receptors (TLRs) are critical sensors for pathogens in the initiation of the innate immunity and for subsequent activation of the adaptive immunity. TLR signaling can be regulated by both positive and negative factors. In vitro studies suggest that heat shock protein (HSP) gp96 in the endoplasmic reticulum is essential for the folding/assembly and the surface expression of TLR1/2/4, but not for IL-1 beta/18 receptor. To understand the function of gp96 in the innate immunity in vivo, we have generated a macrophage-specific gp96 knockout mouse using LysM promoter-driven cre-mediated excision of exon 1 of gp96 gene. gp96-/- macrophage has profound defects in response to the stimulation by all TLR ligands tested. Our results have led us to hypothesize that gp96 is a master immune chaperone for TLRs including intracellular TLRs such as TLR9. In addition, we hypothesize that gp96 plays critical roles for the physiological function of macrophage in dealing with endotoxemia, as well as bacterial infections such as Salmonella and Listeria, both of which are category B pathogens. The first hypotheses will be addressed by the specific aim one , which is focused on elucidating the biochemical, structural and cell biological mechanisms of gp96 in chaperoning TLRs. Aim 2 is designed to understand the significance of macrophage- restricted gp96-TLR interaction (or lack of it) both in vitro and in vivo in the context of sepsis and bacterial infection. Our study on the regulation of TLR folding and the function of macrophage in inflammations, as well as bacterial infections, should have broad implications in areas such as infectious diseases, cancer, and atherosclerosis as well as biodefense against emerging pathogens.